Sertoli cells are the testicular epithelial cells that form the seminiferous tubule and provide the cytoarchitectural support and microenvironment for the developing germ cells (B Jegou 1992 Bailliere's Clin Endocrinol Metab 6:273-311; M K Skinner 1991 Endocr Rev 12:45-77). In a mammalian male testicle, the Sertoli cells are the predominant cells which function to support spermatogenesis by providing a microenvironmental and structural support for the developing germ cells.
Before puberty, the Sertoli cells undergo active proliferation, but at puberty and through adulthood they become a terminally differentiated, post-mitotic cell population. The Sertoli cell number per testis determines the efficiency of spermatogenesis. Sertoli cell function is regulated by the gonadotropin FSH and locally produced paracrine factors. Before puberty, these hormones and growth factors can increase Sertoli cell proliferation, but after puberty they fail to influence cell growth.
The molecular mechanisms involved in the post-mitotic block in post-pubertal Sertoli cells are unknown, but may involve up-regulation of cell cycle inhibitory genes such as p16 and p21. The four known helix-loop-helix Id proteins (Id1, Id2, Id3, and Id4), are considered dominant negative regulators of differentiation pathways, but positive regulators of cellular proliferation. The Id proteins are expressed by post-pubertal Sertoli cells. Similar to other cell systems, Id1, Id2 and Id3 are also transiently induced by serum in Sertoli cells.
Terminal differentiation is the state achieved when a cell exits the cell cycle, to become post-mitotic, and the differentiated gene expression profile allows a specialized function for the cell. Often, these terminally differentiated cells cannot be replenished and once lost can cause abnormal tissue function. Examples of terminally differentiated cells include neurons (Yoshikawa 2000), myocytes (Tam et al. 1995; Wei and Paterson 2001) and Sertoli cells (Walker 2003). Abnormalities or loss of these terminally differentiated cells causes corresponding neuro-degeneration (Jellinger 2003; Turlejski and Djavadian 2002), muscle degeneration (Bicknell et al. 2003) or infertility disease states (Sharpe et al. 2003).
The cellular mechanisms that promote and maintain terminal differentiation are poorly understood. The speculation is that altered signal transduction and cell cycle pathways influenced by unique transcriptional events allow a cell to exit irreversibly the cell cycle and promote a unique spectrum of gene expression required by the cell (Prasad et al. 2003; Wegner 2001; Wei and Paterson 2001; Yoshikawa 2000).
Sertoli cell fate is established in the embryonic gonad at the time of sex determination (Hacker et al. 1995; Lovell-Badge and Hacker 1995) and is followed by a phase of rapid cell proliferation and differentiation. During puberty the final phase of Sertoli cell differentiation occurs which is marked by cessation of proliferation and irreversible changes in Sertoli cell morphology and physiology (Jegou 1992). The changes associated with terminal differentiation of Sertoli cells at puberty, include exit from the cell cycle and the formation of the blood testis barrier. This differentiated phenotype is needed for the proper microenvironment and cytoarchitectural support of the developing spermatogenic cells. The Sertoli cell differentiation is accompanied by the expression of many gene products not present in immature cells such as aromatase, androgen receptor (Bremner et al. 1994), GATA-1 (Ketola et al. 2002), p27kip (Holsberger et al. 2003), SGP-2 (Law and Griswold 1994), and laminin alpha 5 (Schlatt et al. 1996) and transferrin (Norton and Skinner 1992).
In general, hormones and growth factors such as FSH (Simoni et al. 1999), thyroid hormone (Holsberger et al. 2003; Palmero et al. 1995), interleukin-1 alpha (Petersen et al. 2002) and TGF alpha (Petersen et al. 2002), increase proliferation of Sertoli cells obtained from prenatal and pre-pubertal testis. The early post pubertal Sertoli cells also remain responsive to these hormones and growth factors, but fail to proliferate and enter the cell cycle (Buzzard et al. 2003; Sharpe et al. 2003). The molecular mechanisms involved in this switch to a post-mitotic and irreversible exit from the cell cycle at puberty are largely unknown. The altered expression of certain regulatory signaling networks involved in the action of these hormones and growth factors is likely involved (Buzzard et al. 2003; Holsberger et al. 2003). For example, pre-pubertal Sertoli cell proliferation may involve activation of the ERK-MAP kinase pathway and subsequent up-regulation of cyclin D1 in response to FSH (Crepieux et al. 2001). Sertoli cells' exit from the cell cycle may be due to induction of the growth inhibitor p27-CIP1 by TH, T and RA (Holsberger et al. 2003). In addition, FSH may also inhibit post-pubertal proliferation through the activation of the PKA-cAMP pathway (Crepieux et al. 2001). This is consistent with the role of cAMP as an inhibitor of proliferation for many cells (Stork and Schmitt 2002).
The majority of the Sertoli cell functions are regulated by the gonadotropin FSH (Simoni et al. 1999). The loss of FSH actions are reflected in reduced Sertoli cell numbers with no qualitative loss on spermatogenesis, but the total number of sperm are reduced (Krishnamurthy et al. 2000). Previous observations have suggested that quantitative spermatogenesis is dependent on the total number of Sertoli cells established prepubertally (Sharpe et al. 2000). The functions of differentiated Sertoli cell are regulated by a combination of hormones and various growth factors. Optimum cell function is maintained through the activation of various signal transduction pathways including protein kinase A, protein kinase C and calcium mobilization (Braun et al. 2002; Hansson et al. 2000; Jia et al. 1996; Silva et al. 2002). These signal transduction pathways activate a number of transcription factors such as cAMP response element-binding protein (Walker et al. 1995), C/EBPβ (Gronning et al. 1999), c-fos (Norton and Skinner 1992), c-myc (Lim and Hwang 1995), GATA-1 (Yomogida et al. 1994), SF-1 (Hatano et al. 1994), and WIN (Chaudhary et al. 2000). It is postulated that the activation of specific combinations of these transcription factors is in part responsible for stage dependent proliferation and differentiation of Sertoli cells.
Sertoli cells have been shown to express members of the basic helix-loop-helix (bHLH) transcription factor family (Chaudhary et al. 1997; Chaudhary et al. 1999; Chaudhary and Skinner 1999a). The family members of bHLH transcription factors are critical cell-type determinants and play important roles in cellular differentiation. A bHLH domain that is conserved from yeast to mammals characterizes the members of this family (Quong et al. 1993). The bHLH domain consists of two amphipathic helixes separated by a loop that mediates homo- and heterodimerization adjacent to a DNA-binding region rich in basic amino acids (Murre et al. 1994). The bHLH dimers bind to an E Box (CANNTG) DNA consensus sequence present in a wide variety of tissue-specific promoters (Murre et al. 1989a; Murre et al. 1989b). The E box domain has been shown to influence the promoters of a number of Sertoli cell genes, including transferrin (Chaudhary et al. 1997), c-fos (Chaudhary and Skinner 1999b), SF-1 (Daggett et al. 2000), and FSH receptor (Goetz et al. 1996). The bHLH proteins have been classified into two distinct classes. The ubiquitously expressed class A bHLH proteins consist of E2-2 (Bain et al. 1993), HEB (Hu et al. 1992), and E12 and E47 (i.e. differentially spliced products of the E2A gene (Murre et al. 1989b)). The class A bHLH dimerize with tissue-restricted and developmentally regulated class B proteins, such as MyoD and neuroD (Lassar et al. 1991; Massari and Murre 2000). Previous observations suggest that the Sertoli cells express the class A proteins E47 (Chaudhary and Skinner 1999a) and human HEB (Chaudhary et al. 1999) (i.e. the rat isoform of human HEB). Sertoli cell-specific class B bHLH proteins are yet to be determined. However, reports suggest that bHLH proteins regulate FSH-stimulated Sertoli cell gene expression (Chaudhary et al. 1997; Chaudhary and Skinner 1999b; Chaudhary and Skinner 1999c).
The members of the Id (inhibitor of differentiation/DNA binding) family modulate the transcriptional activity of class A and B bHLH heterodimers. The four known Id proteins (Id1, Id2, Id3, and Id4) share a homologous HLH domain, but lack the basic DNA binding region (Benezra et al. 1990; Daggett et al. 2000). Thus, the Id proteins act to sequester bHLH proteins by forming inactive dimers to prevent binding of bHLH proteins to the E-box responsive elements (Einarson and Chao 1995; Langlands et al. 1997; Loveys et al. 1996). Therefore, Id proteins are largely considered as dominant negative regulators of differentiation pathways (Barone et al. 1994; Hara et al. 1994; Moldes et al. 1997), but positive regulators of cellular proliferation. The induction of Id in various cell types has been studied in response to serum, which is known to induce proliferation of most cells.
The biphasic expression pattern of Id1 and Id2 in human diploid fibroblasts after serum stimulation corresponding to G1 phase and G1-S transition supports their role in proliferation (Hara et al. 1994). In addition to Id1 and Id2, Id3 is also induced early after cell cycle stimulation (Christy et al. 1991). The mechanisms by which Id proteins promote the cell cycle are diverse but appear to involve suppression of p21, p27, cyclin A, cyclin E, cyclin dependent kinase-2 (cdK2), and interactions with pRb (Zebedee and Hara 2001). Previous observations suggest that the differentiated Sertoli cells also express Id proteins (Buzzard et al. 2003; Chaudhary et al. 2001; Sablitzky et al. 1998). The functional significance of Id protein expression in terminally differentiated and post-mitotic Sertoli cells is unclear. Recent observations suggest that long-term (72 hour) stimulation of Sertoli cells in culture with FSH down-regulates Id1 and Id3. In contrast, serum up-regulates Id and Id3 expression (Chaudhary et al. 2001). Short-term stimulation of Sertoli cells with FSH (30 min-12 h) up-regulates Id2 in a biphasic manner. This response mimics the effect of mitogens on other cell systems (Zebedee and Hara 2001). The transient up regulation of Id genes in response to FSH suggests that differentiated Sertoli cells may be competent to re-enter the cell cycle if Id gene expression is sustained.
Organ transplantation remains a last-resort treatment for certain diseases that cause chronic organ damage. One of the main obstacles to long-term disease relief is transplant rejection, caused by immune response destruction of the transplanted organ. Presently, the only recourse to combat this immune response is to administer nonspecific immunosuppressive agents (Lancet 345:1321-1325 (1995). Unfortunately, life-long use of immunosuppressive agents increases the risks of cardiovascular disease, infections and malignancies. There remains a need for compositions and methods that decrease transplant rejection in the recipient subject.
One of the diseases that causes chronic organ damage is diabetes mellitus. Organ transplantation has been used to treat diabetes mellitus, but with limited success. Diabetes mellitus is characterized by a relative or complete lack of insulin secretion by the beta cells within the islets of Langerhans of the pancreas, or by defective insulin receptors. A vast number of diabetic patients receiving islet transplants experience transplant rejection and short-term insulin independence. For example, only 12.4% of the patients receiving islet allograft transplants experienced insulin independence for periods of more than one week, and only 8.25% have been insulin independent for periods of more than one year (Linsley et al. 1997 Diabetes 46: 1120-3).
One method for inhibiting transplant rejection includes co-grafting the transplant organ, tissue or cells with non-modified Sertoli cells. It has been previously shown that co-grafting non-modified Sertoli cells with the transplant enhances the viability of transplant organs in the recipient subject (U.S. Pat. Nos. 5,702,700; 5,725,854; 5,759,534; 5,830,460; and 5,849,285). The non-modified Sertoli cells produce a cellular factor, or factors, which create an immuno-privileged site for the co-grafted transplant, thereby enhancing the ability of the transplant to function, mature, proliferate, and/or survive (e.g., enhanced viability). The identity of the factor(s) is not yet known. However, non-modified Sertoli cells are known to produce cellular factors, including IGF (insulin-like growth factor), EGF (epidermal growth factor), and/or transferrin. These factors may or may not inhibit transplant rejection.
Although co-grafting methods using non-modified Sertoli cells offer some relief from transplant rejection, these methods also suffer drawbacks, because the grafted non-modified Sertoli cells do not proliferate and cannot regenerate the tissue. Thus, there still exists a need for cells that inhibit transplant rejection and do not undergo tumorigenesis.
The bHLH proteins are known to mediate cell growth and differentiation. In general, the bHLH proteins form a family of transcription activation factors having a helix-loop-helix domain (C Murre, et al 1994 Biochem Biophys Acta 1218:129-135; C Murre, et al., 1989 Cell 56:777-783) and a DNA binding domain. The helix-loop-helix domain is essential for dimerization with other bHLH proteins. The dimerized protein complex is an activated transcription factor. In the dimerized complex, the DNA-binding domain mediates binding to a consensus E-box (CANNTG) DNA sequence resulting in transcription activation (A B Lassar, et al., 1991 Cell 66:305-315). The E-box sequence is present in various tissue-specific promoters including-specific promoters such as transferrin and others (C Murre, et al., 1989 Cell 56:777-783; C Murre, et al., Cell 58:537-544; J Chaudhary, et al., 1997 Endocrinology 138:667-675; J Chaudhary and M K Skinner 1999 Mol Endocrinol 13:774-786; M A Daggett, et al., 2000 Biol Reprod 62:670-679; T L Goetz, et al., 1996 J Biol Chem 271:33317-33324).
The family of bHLH proteins includes class A and B bHLH heterodimers (M W Quong, et al 1993 Mol Cell Biol 13:792-800). The class A bHLH proteins include E2-2 (G Bain, et al 1993 Mol Cell Biol 13:3522-3529), HEB (J S Hu, et al 1992 Mol Cell Biol 12:1031-1042), E47 (J Chaudhary and M K Skinner 1999 Mol Reprod Dev 52:1-8), REB-alpha (J Chaudhary, et al 1999 Biol Reprod 60:1244-1250), and E12 and E47 which are differentially spliced products of the E2A gene sequence (C Murre, et al 1989 Cell 58:537-544). The class B bHLH proteins include MyoD and neuroD (A B Lassar, et al 1991 Cell 66:305-315; M E Massari and C Murre 2000 Mol Cell Biol 20:429-440).
The Id proteins are also members of the bHLH family of proteins. The Id proteins share the structural helix-loop-helix domains found in the bHLH proteins, however the Id proteins lack the DNA-binding domain. The Id proteins bind the bHLH proteins to form inactive heterodimers, thereby preventing the bHLH proteins from binding their cognate E-box DNA sequences and preventing transcriptional activation by the bHLH protein (M B Einarson and M V Chao 1995 Mol Cell Biol 15:4175-4183; K Langlands, et al. 1997 J Biol Chem 272:19785-19793; D A Loveys, et al., 1996 Nucleic Acids Res 24:2813-2820; M V Barone, et al., 1994 Proc Natl Acad Sci USA 91:4985-4988; E Hara, et al., 1994 J Biol Chem 269:2139-2145; M Moldes, et al., 1997 Mol Cell Biol 17:1796-1804). Thus, the members of the Id protein family modulate transcriptional activity of the bHLH proteins thereby modulating cellular differentiation. The Id2 protein also includes an N-terminal domain which mediates apoptosis.
The bHLH proteins are postulated to interact with other cellular proteins to mediate transcriptional activation of genes involved in cell growth and/or differentiation. It has been suggested that the bHLH proteins activate transcription of gene sequences controlled by an E-box promoter sequence through transcriptional co-activator proteins, such as CREB (e.g., a CRE-binding protein or a cAMP response element-binding protein) (S Suire, et al., 1995 Mol Endocrinol 9:756-766; J Chaudhary and M K Skinner 1999 Endocrinology 140:1262-1271; J Chaudhary and M K Skinner 2001 Biology of Reproduction 65:568-574) and/or through adaptor proteins, such as CBP (cAMP response element-binding protein-binding protein) (J C Chrivia, et al., 1993 Nature 365:855-859; J Chaudhary and M K Skinner 2001 Biology of Reproduction 65:568-574) and/or p300 (R Eckner, et al., 1994 Genes Dev 8:869-884; J Chaudhary and M K Skinner 2001 Biology of Reproduction 65:568-574). Accordingly, the heterologous protein (e.g., Id proteins) expressed in the inventive modified cells may bind the CREB, CBP, and/or p300 proteins to inhibit transcriptional activation, thereby inhibiting cell growth, differentiation, or tumorigenesis of the modified cells.
There still remains a need for cells able to be used as therapeutic agents for treatment of diseases and to inhibit transplant rejection, that do not become tumorigenic in the recipient subject, and as targets in cell based assays for drug discovery and screening.